Treatment of pulmonary and pleural fibrosis using HSP27 inhibitors

ABSTRACT

Reduction of HSP27 expression is in beneficial in the treatment of pleural and pulmonary fibrosis and in particular subpleural fibrosis and IPF. Pharmaceutical compositions for this purpose contain an inhibitor of HSP27 and a pharmaceutically acceptable carrier.

STATEMENT OF RELATED CASE

This application is a continuation of U.S. patent application Ser. No.13/470,780, now U.S. Pat. No. 8,987,223, filed on May 14, 2012, andclaims the benefit of U.S. Provisional Patent Application Ser. No.61/485,501, filed on May 12, 2011, both of which are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

This application related to treatment of pulmonary and pleural fibrosisusing inhibitors as hsp27.

Heat shock proteins (HSPs) are a set of highly conserved proteins whoseexpression is induced by different kinds of stress. Among the differentHSPs, HSP27 is highly induced by different stresses such as hightemperature, oxidative stress, or anticancer drugs (Beckmann et al.,Science 1990; 248:850-4; Shashidharamurthy et al., J Biol Chem 2005;280:5281-9) and is an ATP-independent chaperone. HSP27 is crucial in thecontrol of apoptosis and can act at multiple points in the apoptoticpathways to ensure that stress-induced damage does not inappropriatelytrigger cell death (Bruey et al., Nat Cell Biol 2000; 2:645-52; Jego etal Cancer Lett, Nov. 13, 2010, E Pub ahead of print) probably becauseapoptosis and differentiation are two related processes. Recent reportsinvolve HSP27 in the differentiation process (De Thonel et al., Blood2010, 116:85-96). The cytoprotective function of HSP27 is also explainedby its behavior as a molecular chaperone for other cellular proteins.Inappropriate activation of signaling pathways could occur during acuteor chronic stress as a result of protein misfolding, proteinaggregation, or disruption of regulatory complexes. The action ofchaperones, through their properties in protein homeostasis, is thoughtto restore the balance. In association with HSP27 chaperone function, ithas been demonstrated that HSP27 under stress conditions play a functionin “proteins triage”; HSP27 may either stabilize or accelerate thedegradation by the proteasome of certain “client” proteins to assure thesurvival of the cell (De Thonel et al 2010; Andrieu et al. Oncogene2010, 29: 1883-1896; Parcellier et al., FASEB J 2006, 20: 1179-1181, andParcellier et al., Mol Cell Biol 2003, 23: 5790-5802). The strongcytoprotective function of HSP27 together with the fact that the proteinis overexpressed in most cancer combine to make this chaperone aninteresting target to inactivate in cancer therapy. In this way, HSP27depletion in different animals' models induces the regression of thetumors. The second generation oligonucleotide, OGX-427, is a specificinhibitor of HSP27 that can be administered in patients. Its anti-tumoreffect has been demonstrated in many animal experimental models and itis now being tested in phase I/II clinical trials as a chemosensitizingagent in prostate cancer.

Epithelial-to-mesenchymal transition (EMT) occurs when epithelial cellstransdifferentiate and acquire a myofibroblastic phenotype (Chapman HA., Annu Rev Physiol 2011; 73:413-35) TGF-β1, one of the most potentprofibrotic mediators characterized so far, is also considered to be amaster switch for the induction of EMT in various organs including thelung (Zavadil et al., Oncogene 2005; 24:5764-74). EMT is known to beinvolved in a variety of normal physiological processes includinggastrulation, heart formation, and palate closure during embryogenesisbut also in pathological conditions such as fibrosis, cancer invasionand metastasis. EMT describes a phenotypic change characterized by theloss of epithelial markers including E-cadherin and cytokeratin, thegain of mesenchymal markers such as α-smooth muscle actin (α-SMA), andthe acquisition of cell migration properties. It has been reported thatthe cells migrate across the damaged basement membrane to theinterstitial space, where they become activated myofibroblasts. (Mucsiet al. Acta Physiol Hung 2007; 94:117-31) The presence of α-SMArepresents the most reliable marker of the myofibroblastic phenotype.The mechanisms leading to EMT are still poorly known.

Fibrosis generally is the formation of fibrous tissue as a reparative orreactive process. Pathological fibrosis is the undesirable formation offibrous tissue that is associated with a disease state. In manyinstances, treatment options for fibrosis are presently limited orunavailable. For example, among fibrotic disorders, idiopathic pulmonaryfibrosis (IPF) is a devastating disease characterized by a progressiveand exaggerated extra-cellular matrix (ECM) accumulation and structuralchanges resulting in restrictive impairment of lung functions. Thedisease has no known etiology although some anti-cancerous drugs (e.g.bleomycin) are well known triggering events for the initiation ofpulmonary fibrosis. (www.pneumotox.com. The drug-induced lung diseases.[Website] 2011 July, 2002 [cited 1996]).

The development of IPF is classically thought as an abnormal alveolarrepair and myofibroblasts are the main cellular components responsiblefor the tissue remodeling that occurs during this repair. Thepathological changes in IPF typically start in subpleural lung regions.(American Thoracic Society. Am J Respir Crit Care Med 2000; 161:646-64)This subpleural predominance of fibrosis is even a characteristicfeature of IPF. To date, there is no clear explanation for this. Cool etal. suggest that myofibroblasts form a network, a “fibroblasticreticulum,” extending progressively from the pleura to the underlyingparenchyma. (Cool et al., Am J Respir Crit Care Med 2006; 174:654-8. Werecently reported that over expression of the fibrogenic cytokinetransforming growth factor (TGF)-β1 in the pleural space not onlyinduces progressive pleural fibrosis, but also is associated withabnormal collagen deposition within the subpleural lung parenchyma asobserved in IPF. We demonstrated in this model that mesothelial cellsundergo a transformation similar to what is observed during EMT in thepresence of TGF-β and that these mesenchymal cells migrate and invadethe lung parenchyma, suggesting that mesothelial cells may play a keyrole in IPF (Decologne, et al. J Immunol 2007; 179; 6043-6051).

US Patent Publication No. 2006/0003954 discloses an antisense inhalationtherapy for IPF and other lung diseases in which the antisense istargeted to wild type COL1A.

U.S. Pat. No. 6,887,853 discloses treatment for fibrogenic disease usinggeldanamycin, which is an HSP90 inhibitor. No mention is made of HSP 27.Ryjänen et al. Eur J Pharmacol. 2008 Apr. 28; 584(2-3):229-36. Epub 2008Feb. 14 discloses that geldanamycin increases HSP27 levels. HSP27 isalso known for providing anti-inflammatory properties leading toincreases in IL-10 and IL-12. (See US Patent Application No.2001/004971). Since inflammation may be implicated in IPF and otherforms of fibrosis, (See, Wilson et al, Mucosal Immunol. 2009 March;2(2): 103-121), this combination of teachings would seem to suggest thatreduction of HSP27 would be ill-advised in the treatment of fibrosis,including IPF.

SUMMARY OF THE INVENTION

It has been surprisingly found that reduction of HSP27 expression is infact beneficial in the treatment of pulmonary fibrosis and in particularsubpleural fibrosis and IPF. Thus, in accordance with a first aspect ofthe present invention there is provided a method for treating pulmonaryfibrosis comprising administering to an individual in need of treatmentfor pulmonary fibrosis an inhibitor of HSP27.

In a further aspect, the present invention provides a pharmaceuticalcomposition for treatment of pleural or pulmonary fibrosis comprising aninhibitor of HSP27 and a pharmaceutically acceptable carrier. Thiscomposition may be for use in the treatment of idiopathic pulmonaryfibrosis (IPF); or any one of pleural fibrosis, subpleural fibrosis,pulmonary fibrosis, Usual Interstitial Pneumonia (UIP) or drug-inducedlung fibroses.

The inhibitor in the pharmaceutical composition is suitably selectedfrom the group consisting of nucleotide inhibitors, peptide and antibodyinhibitors, and small molecule inhibitors, for example a nucleotideinhibitor of HSP27 such as Seq ID No. 2.

The pharmaceutical composition is suitably configured for administrationby way of inhalation; or by way of any one of intra-pleural injection,intravenous injection or intra-tracheal administration and the form ofthe composition and pharmaceutically acceptable are selected for thispurpose. One specific suitable carrier comprises phosphate bufferedsaline.

The invention further encompasses use of an inhibitor of HSP27 or apharmaceutical composition comprising an inhibitor of HSP27 and apharmaceutically acceptable carrier in the treatment of pleural orpulmonary fibrosis and a method of such treatment. Such use of methodmay be for the treatment of idiopathic pulmonary fibrosis (IPF); or anyone of pleural fibrosis, subpleural fibrosis, pulmonary fibrosis, UsualInterstitial Pneumonia (UIP) or drug-induced lung fibroses.

The invention also encompasses the use of an inhibitor of HSP27 in themanufacture of a medicament for the treatment of pleural or pulmonaryfibrosis as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the increased expression of HSP27 following infection oflung cells with a vector encoding TGF-β1 (AdTGF-β1)

FIG. 1B shows the increased expression of HSP27 following intra-trachealadministration of bleomycin to mice. Western blot was performed on wholelung protein extracts from mice receiving during 21 days either NaCl orBleomycin. HSC70 is used as a loading control, and the amounts wereevaluated by densitometry.

FIGS. 2A-C show mRNA levels for HSP27, α-SMA, and E-cadherin,respectively, after treatment of human mesothelial Met-5A cells withrTGF-β1 at a concentration of 10/ng/ml for 48 h as compared to untreatedcontrols. RNA expression levels were analyzed by PCR. Bars, SD (n=4).

FIG. 2D-F protein levels for HSP27, α-SMA, and E-cadherin, respectively,after treatment of human mesothelial Met-5A cells with rTGF-β1 at aconcentration of 10/ng/ml for 48 h as compared to untreated controls.RNA expression levels were analyzed by PCR. Bars, SD (n=4).

FIGS. 3A-C show mRNA levels for α-SMA, E-cadherin and PAI-1,respectively, after treatment with a scrambled siRNA control, anti-HSP27siRNA, scrambled siRNA control plus rTGF-β1, or anti-HSP27 siRNA plusrTGF-β1 ((10 ng/ml, 48 h)). Bars, SD (n=4).

FIGS. 3D-F show protein levels for α-SMA, E-cadherin and PAI-1,respectively, after treatment with a scrambled siRNA control, anti-HSP27siRNA, scrambled siRNA control plus rTGF-β1, or anti-HSP27 siRNA plusrTGF-β1 ((10 ng/ml, 48 h)). Bars, SD (n=4).

FIG. 3G shows relative expression of HSP27 (based on HSC70 expression)after treatment with TGF-β1 alone and with si-RNA targeting hsp27 or ascrambled control.

FIG. 4A shows ELISA measurement results of active TGF-β1 in PLF fromrats that have received 6 days treatment by intra-pleural administrationeither an adenovirus control (AdDl) or an adenovirus encoding TGF-β1(AdTGF-β1) together with an ASO control or OGX-427 (12 mg/kg). Bars, SD,n=9.

FIG. 4B compares Hsp27 protein expression in the pleural liquid of micetreated with AdTDF-β1 and a control antisense, with that in mice treatedwith AdTDF-β1 and OGX-427

FIG. 5A shows the mean volume of RCL2 (fixation solution) necessary toinsufflate the lungs in rats receiving either AdDL or AdTGF-β1 and/orOGX427 or an ASO control. 10 ml are necessary to insufflate a healthylung. **p≦0.01, n=6.

FIG. 5B shows quantification of pleural collagen as determined frommicroscopic images.

FIG. 6 shows quantification of collagen in parenchymal subpleural areas.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have determined, as exemplified below, that HSP27is involved in pulmonary and pleural fibrosis, and that targeting HSP27in vitro or in vivo inhibits TGF-β1-induced fibrosis. The presentinventors have also shown that HSP27 expression is linked to endothelialto mesenchymal and mesothelial to-fibroblastoid transitions((collectively EMT), and that mesothelial cells are highly susceptibleto undergoing EMT suggesting they are very aggressive and sensitivecells in the process of EMT and therefore of pleural and pulmonaryfibrosis. Furthermore, in IPF lung tissue, HSP27 is expressed at highlevel in clusters of bronchiolar basal cells precisely localised withinfibroblast foci, wedged between luminal epithelial cells andmyofibroblasts where EMT is supposed to occur. (Chilosi et al. RespirRes 2006; 7:95; Korfei et al., J Proteome Res. 2011, Mar. 29 E Pub aheadof print). Our work here shows that HSP27 is overexpressed duringpleural/pulmonary fibrosis development most probably though itsimplication in TGF-β1-induced EMT. Further, we demonstrate that HSP27overexpression on its own is sufficient to induce EMT. Interestingly,the function of HSP27 does not seem restricted to mesothelial cellsundergoing EMT but most probably also concerns epithelial cells as wedemonstrate in A549 cells.

Taken together, these results support the validity of the presentinvention, in which reduction of HSP27 is used in the treatment ofpulmonary and pleural fibrosis.

DEFINITIONS

The term “HSP27” refers to heat shock protein 27, an approximately 27kilodalton stress-induced protein. HSP27 is also sometimes referred toas heat shock protein beta-1 (HSPB1). The sequences of HSP27 are knownin the art for Homo sapiens (AB020027, X54079, NM_006308, NM_001540 andNM_001541), dogs (NP_001003295), cattle (NP_001020740), mice (NP_038588)and other species. A sequence for human HSP27 is provided as Seq ID No.1.

The term “inhibitor” or “inhibition” of HSP27 refers to a composition oraction that leads to a reduction in the amount of active HSP27. Thereduction may occur at the expression level, or throughpost-expression/post-translation inactivation of HSP27. Examples ofinhibitors includes nucleotide compounds targeting HSP27, peptideaptamers (Gilbert et al, Oncogene. 2011 Mar. 21. [Epub ahead of print]);flavonoid inhibitors of HSP27, antibodies that interact with HSP27(Tezel and Wax, J. Neuroscience 10:3553-3562 (2000), and interferon-γwhich has been shown to downregulate expression of HSP27. (Yonekura etal., (2003) Cell Death and Differentiation 10, 313-322).

The terms “treatment” or “treating” refer to the administration or useof an HSP27 inhibitor for the purpose of obtaining a therapeutic benefitin an individual in need for treatment for pulmonary fibrosis. Thetherapeutic benefit may be a reduction in the severity of existingfibrotic condition, a slowing of the progression of an existing fibroticcondition or the delay in onset of a potential fibrotic condition. Theterm ‘therapeutically effective amount” refers to an amount that isreasonably expected to achieve such a therapeutic benefit, even if nobenefit is discernable in a particular treated individual.

The term “individual” refers to a living mammal being treated. Inspecific embodiments, the individual is a human, although treatment inother mammalian species, particularly pet and agricultural species suchas dogs and cattle is within the scope of the invention.

The term “pleural and pulmonary fibrosis” refers to pathologicalfibrosis of the lungs whether arising from disease or injury. The termencompasses IPF, pleural fibrosis, subpleural fibrosis, pulmonaryfibrosis, Usual Interstitial Pneumonia (UIP) and drug-induced lungfibroses.

Inhibitors of HSP27

In the methods and pharmaceutical compositions of the invention, aninhibitor of HSP27 is used.

In accordance with some embodiments, the HSP27 inhibitor is a nucleotideinhibitor. Examples of nucleotide inhibitors include antisensesequences, which may be full-length antisense (see Horman et al., Int.J. Cancer (1999) 82: 574-582), or shorter oligonucleotide sequences,having a length of 100 bases or less, for example 12 to 30 bases. Suchantisense species are complementary to the target HSP27 gene to anextent sufficient to achieve antisense inhibition in vivo, and mayinclude degeneracy to take into account allelic variation. Specificoligonucleotide antisense inhibitors of HSP27 are known in the art fromUS Patent Publications 2004/0127441, 2009/0264502

In specific embodiments, the HSP27 inhibitor is OGX-427, an antisenseoligonucleotide made by OncoGenex that is currently in clinical trialsfor treatment of various types of cancer. OGX-427 is a 4-12-4 2′-MOEgapmer oligonucleotide with phosphorothiolated internucleotide linkageswhich can be represented as

(Seq. ID No. 2) 5′-GGGAMeCGMeCGGMeCGMeCTMeCGGMeUMeCAMeU-3′where G, A, MeC, and T represent the nucleosides 2′-deoxyguanosine,2′-deoxyadenosine, 2′-deoxy-5-methylcytidine, and 2′-deoxythymidine, theunderlined nucleosides denote 2′-O-methoxyethyl (2′-MOE) modificationsof the nucleosides, and the internucleotide linkages are phosphothioatediester, sodium salts.

In other embodiments, the nucleotide HSP27 inhibitor is a doublestranded RNA species (or precursor) that operates by an siRNA mechanismto reduce expression of HSP27. Specific RNA species for this purpose areknown from US Patent Publication 2004/0127441, and Chauhan, et al.(2003) Cancer Res. 63, 6174-6177.

Peptide aptamers (Gilbert et al, Oncogene. 2011 Mar. 21. [Epub ahead ofprint]); and antibodies (Tezel and Wax, J. Neuroscience 10:3553-3562(2000) that interact with HSP27 are also known and could serve asinhibitors of HSP27 in accordance with the invention. Other peptidesthat bind to HSP27, such as CP91 or binding fragments thereof asdescribed in US Patent Publication No. 2007/0003555 could also beemployed.

Cytokines such as interferon-γ are also known to inhibit HSP27 and canbe used as inhibitors in the present invention. Yonekura et al., CellDeath and Differentiation (2003) 10: 313-322.

Other inhibitors of HSP27 are also known which are generally “smallmolecule” inhibitors. These include flavonoids such as quercetin,(Morino et al., in vivo (1997) 11: 265-270; JP 10045572, JP 10045574,JP10036261 and JP 10036267), and biphenyl isooxazoles such as5-(5-Ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)isoxazole(KRIBB3) (Shin et al. The Journal of Biological Chemistry VOL. 280, NO.50, pp. 41439-41448, Dec. 16, 2005). KRIBB3 is available commerciallyfrom Sigma-Aldrich is understood to reduce HSP27 activity by acting as aspecific inhibitor (IC₅₀ of 50 nM) of PKC-dependent phosphorylation ofHSP27. Berberine derivatives have also been shown to inhibit hsp27. (EP0 813 872) Paclitaxel has also been shown to be an inhibitor of hsp27expression. (Tanaka et al., Int J Gynecol Cancer. 2004 July-August;14(4): 616-20).

Administration and Formulation

The methods of the present invention involve administration of an HP27inhibitor as described above to an individual in need of treatment, orthe incorporation of the inhibitor into a pharmaceutical compositionsuitable for administration. As will be appreciated, the mode ofadministration and the pharmaceutical carrier employed will varydepending on the specific inhibitor and the condition for which thetreatment is intended.

In general, administration may be by any known approach includingintravenous, oral, intramuscular, intranasal, or inhaled. In specificembodiments, administration is suitably regional to the area to betreated, i.e. the lungs. For example, inhalation is one mode of regionaladministration. Inhalation strategies for antisense therapeutics areknown, for example from Karras et al, Drug Discovery Today: TherapeuticStrategies (2006) 3(3): 335-341 and Crosby et al, J Pharmacol. and Exp.Therapeutics. (2007) 321: 938-946. See also, US Patent Publication No.2006/0003954. Other modes of regional administration to the lungsinclude intra-pleural injection and intra-tracheal administration.

In one method, the HSP27 inhibitor is an antisense oligonucleotide. Onesuch product is a compound called “OGX-427” which is currently providedto patients at about 600 mg per patient in a 25 mg/mL concentrationformulated as a mannitol-phosphate buffer solution (pH 7.4) for IVadministration. OGX-427 dosing solutions are administered intravenouslyusing an infusion pump. In some situations, the administration will bepreceded or accompanied by administration of an antihistamine.

Experimental Results Materials and Methods Recombinant Adenovirus andAntisense Oligonucleotides

For experiments on pleural fibrosis we used AdTGF-β1223/225, anadenovirus construct with a mutant TGF-β1 translated into spontaneouslybioactive TGF-β1 (AdTGFβ1) and control vectors (AdDL) with no insert inthe deleted E1 region. The construction of adenoviral vectors isdescribed in detail elsewhere (DeCologne, 2007, supra; Bett et al. ProcNatl Acad Sci USA 1994; 91:8802-6; Sime et al., J Clin Invest 1997;100:768-76)

OGX427 (SEQ ID No: 2), a second-generation AntiSense Oligonucleotidstargeting Hsp27, was provided by OncoGeneX (Vancouver Canada). A controloligonucleotide having the sequence CCTTCCCTGAAGGTTCCTCC (SEQ ID No: 3)was obtained from Martin Gleave at the Prostate Cancer Center, VancouverCanada and is also known as ISIS 141923.

Animal Treatment

Female Sprague-Dawley rats (Charles River Laboratories) weighing 200-225g were housed in special pathogen-free conditions. Rodent laboratoryfood and water were provided ad libitum. The animals were treated inaccordance to the guidelines of the Ministère de la Recherche et delaTechnologie (Paris, France). All experiments were approved by theComitéd'Ethique de l'Universite´ de Bourgogne (Bourgogne, France). Allanimal procedures were performed with inhalation anesthesia withisoflurane (TEM, Lormont, France). Adenovector administrations wereperformed by intrapleural injection in 800 μl of NaCl0.9% containing atotal of 1.3×10⁹ PFU AdTGF-β1, AdDL or AdLacZ. The intrapleuralinjection protocol was described in details elsewhere (DeCologne, 2007,supra)).

12 mg/kg of control ASO and OGX-427 were daily administrated byintrapleural injection during 5 days after adenoviral administration.Rats were euthanized by abdominal aortic bleeding and lungs wereharvested and processed following what was previously described inDecologne et al (DeCologne, 2007, supra)).

For primary cell harvesting, 8 ml of HBSS (Lonza, Paris, France) wereinjected in the pleural cavity through the diaphragm then removed andfollowed by a 10 ml trypsin 5%, EDTA 0.4% administration for 30 min.This lavage was removed and placed in 30 ml of DMEM (Dulbecco's ModifiedEagle Medium, Lonza) with 15% FBS (Fetal Bovine Serum, Lonza). Aftercentrifugation (1300 rpm, 5 minutes), the cell pellet was re-suspendedinto 5 ml DMEM, FBS 15% and placed at 37° C., 5% CO₂.

For lung tissue or histological analysis after intratrachealadministration of bleomycin in mice or AdTGF-β1 in rats we used materialfrom previous experiments (respectively Decologne et al, Eur Respir J2010; 35:176-85, and Bonniaud et al. Am J Respir Crit Care Med 2005;171: 889-98).

Histology

Immunohistochemitry: Inflated lungs were transversally cut and paraffinembedded. 5 μm section were performed and stain with primary antibodies(Ab), Mouse monoclonal anti-α-SMA (Sigma, St Quentin Fallavier, France,1/200), rabbit polyclonal anti-HSP27 (EnzoLife Science, Villeurbanne,France, 1/200) and rabbit polyclonal cleaved caspase 3 (cell signaling,Saint Quentin, France, 1/100). Secondary Abs used were respectively, agoat anti-mouse IgG biotin conjugated and a goat anti-rabbit IgG biotinconjugated. After peroxidase inhibition (PBS plus H2O2, 20 min), lungsection were incubated over night at 4° C. with primary ab. Tissuesection were after PBS 1× (Phosphate Buffer Saline) wash incubated withthe secondary ab (1/500) 45 min. The streptavidin-HRP complex (JacksonImmunoResearch Laboratories, PA, USA, 1/500) was used 45 min at roomtemperature. 3-Amino-9-ethylcarbazole/hydrogen peroxide was used aschromagen substrate. Slides were counterstained with hematoxylin.

Cytochemical staining for β-galactosidase was performed as described inDeCologne, 2007, supra.

Determination of TGF-β1 Level in PLF

Total human TGF-β1 was determined from pleural lavage fluid (PLF) usingELISA (R&D Systems, Lille, France), performed according to therecommendations of the manufacturer. The sensitivity of this assay was 7pg·mL-1.

Cell Culture

Mesothelial rat primary cells were grown in Dulbecco's Modified Eagle'sMedium (DMEM) 1% L-glutamine, 1% penicillin-streptomycin with 15% FetalBovine Serum (FBS. Lonza, Paris, France). Cells were assessed beforeculture and from passage 1 to 4.

Mesothelial rat established cell line 4/4 RM4 cells (American TypeCulture Collection—ATCC—Rockville, Md.) were seeded in Ham's F10(Lonza), with FBS 15%. Mesothelial human established cell line MET-5A(ATCC) were seeded in Medium 199 (lonza), 1% L-glutamine, 1%penicillin-streptomycin with 10% FBS. Epithelial human cell line A549cells (ATCC) and were seeded in Ham's F10 (lonza) with 10% FBS.

For EMT induction and analysis, all the different cells were exposed totheir respective medium containing 10 ng/ml recombinant (r)TGF-β1 (R&Dsystem, Lille, France) for 6, 12, 24, or 48 hours.

RNA Interference

HSP27 siRNA were purchased from applied bios system (Courtaboeuf,France). The sense and antisense sequence were respectively:5′-GUUCAAAGCAACCACCUGUtt-3 (SEQ ID No: 4)′ and5′-ACAGGUGGUUGCUUUGAACtt-3′. (SEQ ID No: 5). They were used at aconcentration of 50 nM. SiRNA were transfected using INTERFERin(polyplus, Illkirch, France) following the manufacturer recommendations.5-6 hours after transfection medium were changed and cells were treatedor not with 10 ng/ml TGF-β1 for 12, 24 or 48 hours. Seq ID No. 3 wasused as a random control oligonucleotide.

Transfection

HSP27 transfection was made using a pcDNA6 plasmid encoding human HSP27as previously described. (Bruey et al. Nat Cell Biol 2000; 2:645-52)Transfections were performed using OptiMEM (Invitrogen, Cergy pontoise,france) media and Nanojuice transfections reagent (Novagen, Merck KGaA,Darmstadt, Germany). Cells were lyzed 6, 9, 12, 24 or 48 h aftertransfections.

Heat Shock

Cells were placed in 42° C. water for 30 min and placed back at 37° C.,5% CO₂, 6 hours before protein or RNA analysis.

Western Blot

Protein extraction was made using triton X 100 during 30 min at 4° C.,vortexing every 10 min. Protein dosage is made with the modified lowrykit (Biorad, Marne-la-Coquette, France). Equal amounts of protein (30-50μg) were electrophoresed through 8 to 12% SDS polyacrylamide gels. Theseparate protein bands were transferred onto a PVDF membrane. Thenon-specific sites were saturated with a solution of PBS-tween 0.1%,nonfat milk 8%. Membranes were incubated with primary Abs over night at4° C. Secondary Abs IgG linked to HRP (HorseRadish Peroxidase, JacksonImmunoResearch Laboratories, PA, USA) were used at a concentration of1/5000. Enhanced ChemiLuminescent substrat (ECL, santa cruzbiotechnology, CA, USA) was used to detect the protein of interest.Primary Abs were ?-SMA mouse monoclonal Ab (Progen, 1/1000), a HSP27Rabbit polyclonal Ab (Enzo life science, Villeurbanne, France; 1/2000),E-cadherin mouse monoclonal Ab (R&D systems, 1/1000), PAI-1 was mousemonoclonal Ab (thermo scientific, 1/1000), SMAD2, phosphoSMAD3, TGF-?1were rabbit polyclonal Abs (cell signaling, Saint Quentin, France,1/1000), SMAD3 was rabbit polyclonal Ab (thermo fisher, Illkirch,France; 1/1000), HSC70 was a mouse monoclonal ab (santa cruzbiotechnology, CA, USA; 1/10000), rhoA was mouse monoclonal Ab(cytoskeleton, Denver, USA; 1/1000), calretinin was a mouse monoclonalAb (santa cruz biotechnology, CA, USA; 1/1000), cytokeratin was a mousemonoclonal Ab (affinity, 1/1000).

Co-Immunoprecipitation

Santa Cruz Protein G-sepharose were incubated at 4° C. on well withHSP27 polyclonal Ab during 90 min. Extracted proteins from Met-5A orA549 were add to the sepharose/Ab mix and incubated on well over nightat 4° C. The mixture was centrifuged 5 min and the resulting pellet waswashed three times with the lysis solution. Western blots were performedon the immunoprecipitated proteins.

Immunofluorescence

Cells were fixed with ParaFormaldehyde (PFA) 4% solution andpermeabilized with PBS-triton 0.1% solution. Saturation of non specificsites was made with BSA 5%. Cells were incubated with primary Abs at1/200 over night in humid chamber at 4° C. Cells were stained for α-SMA(Sigma, 1/200), HSP27 (Enzo life science, Villeurbanne, France; 1/200),E-cadherin (BD System, Le Pont-de-Claix, France, 1/100), Calretinine(santa cruz technology, CA, USA; 1/200), F-actin (phalloidin, 1/300).

As secondary Abs Alexa-488 and Alexa-568 conjugated goat anti-mouse orgoat anti-rabbit were used at 1/2000. DAPI was used to stain nucleus.Photos were taken with ×64 objective (Zeiss, Le Pecq, France).

Quantitative RT-PCR

Total RNA was extracted using TRIzol (Invitrogen, Cergy Pontoise,France) from MET-5A and A549 cells treated or not with rTGF-β1 12 or 24hours and transfected or not by HSP27 siRNA or plasmid encoding HSP27.Reverse transcription was performed on the total RNA using the M-MLV kit(Promega, Charbonnieres, France). Quantitative RT-PCR was performed onthe cDNA using the SYBR green master mix (promega, Charbonnieres,France). The sense and antisense primers for HSP27 were5′-GCCCCCATGCCCAAGCT-3′ (SEQ ID No. 6) and 5′-CTCGAAGGTGACTGGGATGGT-3′(SEQ ID NO: 7), respectively. The sense and antisense primers for α-SMAwere 5′-TGGTCGGTATGGGTCAGAAAG-3′ (SEQ ID No: 8) and5′-TCAGGGTCAGGATACCTCTCTTG-3′ (SEQ ID No: 9), respectively. The senseand antisense primers for E-cadherin were 5′-ACAGCCCCGCCTTATGATT-3′ (SeqID No. 10) and 5′-CTTCGGAACCGCTTCCTTCA-3′ (Seq ID No. 11), respectively.The sense and antisense primers for PAI-1 were5′-CGTGGTTTTCTCACCCTATGG-3′ (Seq ID No. 12) and5′-CTGGGTTTCTCCTCCTGTTGTC-3′ (Seq ID No: 13) respectively.

Statistical Analysis

Comparisons between groups were performed by the Mann-Whitney test andcomparisons between animals in the same group by the Wilcoxon test.

Results HSP27 is Overexpressed During Lung Fibrosis In Vivo

Rats receiving an adenovirus encoding for TGF-β1 (AdTGF-β1) byintrapleural or intratracheal injection developed, as previouslypublished, pleural/subpleural (DeCologne, 2007, supra) and parenchymalfibrosis (Sime et al, supra), respectively. We demonstrated here byimmunohistochemistry and western blot analysis of pulmonary tissue thatHSP27 together with the well-known mesenchymal/fibrosis marker, α-SMAwere strongly expressed in animals that have been treated with AdTGF-β1and that developed a fibrosis. Both proteins co-localized in fibroticareas. In contrast, hardly any expression of α-SMA and HSP27 wasobserved in the control animals that received an empty adenovirus AdDLand that did not develop a fibrosis. These results were confirmed inanother pulmonary fibrosis model. Intra-tracheal administration ofbleomycin in mice is a classical animal model of fibrosis thatreproduces the acute lung injury found in patients. We observed thatparalleling fibrosis formation, bleomycin intra-tracheal administrationinduced a significant HSP27 overexpression which was concomitant withthe induction of the mesenchymal marker α-SMA, as assessed byimmunoblotting in animals' lung tissue (FIGS. 1A and B)

HSP27 is a well known anti-apoptotic protein (Bruey et al, Nat CellBiol). However, HSP27 overexpression in this fibrotic context could notbe associated with apoptosis since the small amount of apoptoticmesothelial and epithelial cells found was identical in lung tissuesoverexpressing HSP27 and developing a fibrosis than in those fromcontrol animals that do not develop a fibrosis and with a HSP27expression very weak. Thus, the overexpression of HSP27 seemed relatedto other fibrotic triggering events.

Mesothelial Cells are Prone to Undergo EMT In Vitro

We previously demonstrated that during fibrosis induced by adenoviralTGF-β1 infection in rodents, pleural mesothelial cells were able totrans-differentiate and acquire myofibroblast proprieties that includedα-SMA expression and the ability to migrate into the pulmonaryparenchyma. This particular process in which the starting cells were notepithelial but mesenchymal, seemed essential for pleural and pulmonaryfibrosis. To study whether we could reproduce in vitro this process, wecultivated human Met-5A and rat 4/4 RM4 mesothelial cells in thepresence of recombinant TGF-β1. These cells were able to go in culturethrough this process as demonstrated, both at the RNA and protein level,by the increase in α-SMA expression (FIGS. 2B and 2E) and theconcomitant loss in E-cadherin expression (FIGS. 2C and 2F).

We next confirmed this in vitro process in primary mesothelial cellsextracted from rats. In contrast to the established mesothelial celllines, primary mesothelial cells did not need rTGF-β1 to undergo EMT,but they seem to “trans-differentiate” spontaneously as they wentthrough culture passages. Immediately after extraction from the rats,primary mesothelial cells expressed epithelial markers such ascytokeratin or E-cadherin and the mesothelial marker calretinin, butthey did not express the mesenchymal marker α-SMA. After the firstpassage, these primary mesothelial cells started to express α-SMA. Aftertwo passages, they did not express cytokeratin anymore whereas α-SMAexpression progressively increased over passages and that of E-cadherindecreased. Moreover, while at passage number 1 mesothelial cells had theexpected flattened ovoid shape, at passage number 4 they acquired astrong fibroblastoïd like shape. The mesothelial marker calretinin wasused as a control for the absence of fibroblasts contamination in theculture. All together, we provide evidence that mesothelial cellsundergo EMT in vitro.

HSP27 Expression Increases During EMT as Myofibroblats Features Appearand Associates with α-SMA

Because HSP27 was overexpressed in fibrotic areas in vivo and EMT isimportant in the fibrotic process, we hypothesized that HSP27 may play arole in the process of EMT. We next studied HSP27 expression during theabove mentioned EMT process in vitro. We found, both in primarymesothelial cells and in the human mesothelial cell lines undergoingEMT, that HSP27 expression increased, at the RNA (FIG. 2C) and proteinlevel, as the cells started expressing the myofibroblast marker α-SMA.Immunofluorescence experiments demonstrate that both HSP27 and α-SMAcolocalized during rTGF-β1-induced EMT in human cultured mesothelialcells and that these two proteins associated. These results wereconfirmed in vivo in our fibrosis model. Immunofluorescence andimmunohistochemistry staining showed that HSP27 and α-SMA colocalized infibrotic areas during pleural and pulmonary fibrosis.

This HSP27 overexpression was not just specific of mesothelial cellsundergoing EMT but was also found during the classical EMT involvingepithelial cells. HSP27 was found to be overexpressed, coincidentallywith the expression of α-SMA, in epithelial A549 cells duringrTGF-β1-induced EMT. (Kasai et al. Respir Res 2005; 6:56) Thus, duringTGF-β1-induced EMT, HSP27 was overexpressed paralleling the appearanceof α-SMA with which it associated.

HSP27 Overexpression Induces an EMT Process in Mesothelial CellsProbably Through TGF-β1 Induction and α-SMA Stabilization

To study the involvement of HSP27 in the EMT process, we induced theoverexpression of HSP27 in the human mesothelial Met-5A cells by threedifferent methods: heat shock, staurosporine treatment, and transfectionwith a plasmid encoding HSP27. As expected, a heat shock (30 min at 42°C. followed by 6 h incubation at 37° C. to allow HSP expression) and acytotoxic agent like staurosporine were efficient inducers of HSP27.(Brunet Simioni et al., Oncogene 2009; 28:3332-44) A more specificoverexpression was obtained by HSP27 cDNA transfection. Whatever themethod used, HSP27 overexpression resulted in an increase in α-SMAexpression/content. Conversely, siRNA-meditated depletion of HSP27inhibits rTGF-β1 induced α-SMA in human endothelial Met-5A cells andepithelial A549 cells. This decrease in α-SMA content induced by HSP27depletion seems to be, at least in part, the result of its proteasomaldegradation because in the presence of an inhibitor of the proteasome,such as MG132, α-SMA levels were restored

Transfection-induced HSP27 overexpression was not only able to increasethe content in α-SMA but also reproduced other EMT features induced byTGF-β1. HSP27 gene transfer (without any rTGF-β1) was associated with adecrease in E-cadherin expression and, immunofluorescence stainingshowed that HSP27 overexpression leads to the formation of α-SMA fiberswith an even stronger organization than that induced by TGF-β1treatment. In contrast, depletion of HSP27 by means of a specific siRNAblocks rTGF-β1-induced E-cadherin down-regulation and α-SMAupregulation.

We next further compared EMT induced by HSP27 overexpression with thatinduced by TGF-β1. In mesothelial Met-5a cells and in A549 epithelialcells, transfection-induced HSP27 overexpression was able to induceTGF-β1 and, thereby, we also observed an increase in the proteins thatparticipate in TGF-β1 signal transducing pathway (i.e. Smad2 and Smad3and the anchor protein SARA). Moreover, genes whose expression is knownto be modulated by TGF-β31, like α-SMA and PAI-1 (upregulated by TGF-β1)or E-cadherin (downregulated by TGF-β1) seem identically modulated byHSP27 overexpression (FIGS. 3A to 3F). Altogether, we conclude thatHSP27 overexpression might induce an EMT in mesothelial cells throughits effect inducing the production of TGF-β1 and, most probably, by itschaperon function stabilizing α-SMA protein.

HSP27 Inhibition Blocks EMT in Cultured Cells

To further demonstrate the inhibitory role of HSP27 depletion in thisEMT process, we performed a scratch test on mesothelial Met-5A cells invitro to analyze the acquisition of a migration capacity after treatmentwith rTGF-β1. Cells treated with rTGF-β1 alone and with rTGF-β1/scramblesiRNA had a strong migration capacity and the wound was totallyrecovered by cells within 48 hours. In contrast, untreated cells orHSP27 siRNA cells treated with rTGF-β1 presented a much weaker migrationand, by 48 h, the wound was still largely unrecovered. Therefore, HSP27inhibition blocked this all well known features of EMT induced by TGF-β1in cultured cells. This corresponded to HSP27 expression levels (FIG.3G).

Next, we tested the second generation antisense oligonucleotide OGX-427.Primary mesothelial cells treated with an antisense oligonucleotide(ASO) control were, as expected, able to spontaneous go through EMT withthe successive passages in vitro as demonstrated by the increase inα-SMA and HSP27 expression. In sharp contrast, in the cells receivingOGX427, the increased expression of HSP27 and α-SMA observed with thecell passages was blocked. Moreover, cells receiving OGX427 kept theirmesothelial phenotypic shape in contrast to control ASO treated cells,further proving the EMT blockage induced by OGX-427. A similarinhibitory effect for OGX-427 was observed in human mesothelial MET-5Acells in which EMT was induced by rTGF-β1.

Inhibition of HSP27 by OGX-427 Block Fibrosis In Vivo

To assess the role of HSP27 inhibition in the process of fibrogenesis invivo, we administered OGX427 or its control ASO in the pleural cavity inour AdTGF-β1 induced pleural and subpleural fibrosis model in rats. Theconcentrations of ASO control and OGX-427 used were those previouslydescribed in studies of bladder cancer where the oligonucleotides wereadministered intra-vesically. (Matsui et al. Mol Cancer Ther 2009;8:2402-11)

After AdTGF-β1 or AdDL intrapleural administration, rats were dailytreated with the ASO control or OGX427 during 6 days by intra pleuralinjection. We first confirmed by an ELISA technique that the content onTGF-β1 after AdTGF-β1 intrapleural administration was similar in thepleural lavage fluid (PLF) from all rats, those treated with OGX427 andthose treated with a control ASO (FIG. 4A). A western blot on PLFconfirmed that intra-pleural administration of OGX427 strongly blockedHSP27 expression. (FIG. 4B)

To determine lung activity, lung volumes were assessed by measuring thevolume of RCL2 (fixation solution) that drained into the lungs after 10min at a constant pressure of 20 cm H20. Compared to AdDL administeredanimals, AdTGF-β1 administration in ASO control rats induced a reductionin the lungs volume of 55%. Only a reduction of 35% was observed inOGX427 treated lungs (p<0.01 between ASO and OGX427. FIG. 5). Thiseffect for the HSP27 inhibitor was only observed in the TGF-β1-exposedanimals. In AdDL-administered animals, no difference in lung volumes wasobserved between ASO control- or OGX 427-treated animals (FIG. 5A). Ahistological evaluation of the lungs from rats treated with AdDLdemonstrated that, consistent with other observation, those rats did notpresent any pleural/pulmonary fibrosis. Also, rats treated with AdTGF-β1and receiving the control ASO had major pleural fibrosis with strongHSP27 and α-SMA overexpression. In contrast, rat lungs treated withAdTGF-β1 and receiving OGX427 had minor pleural fibrosis with a verysignificant decrease in HSP27 and α-SMA expression. FIG. 5B showsquantification of pleural coallagen based on microscopic images anddemonstrates the substantial reduction in collagen in animals treatedwith OGX-427 when compared to those treated with AdTGF-β1 and a controlantisense.

In a previous report where we co-administrated AdTGF-β1 together withAdLacZ, we demonstrated that during fibrosis development, mesothelialcells progressively migrated into the pulmonary parenchyma (DeCologne,2007, supra). This AdTGFβ-1 induced migration of mesothelial cellsexpressing AdLacZ (blue cells) is not modified by the daily intrapleuraladministration of control ASO. In contrast, in OGX427 treated animals,the morphology of the cells did not change and no mesothelial cellmigration within the subpleural parenchyma was observed. FIG. 6 showsquantification of collagen in parenchymal subpleural areas. We concludethat HSP27 inhibition by OGX-427 blocks mesothelial EMT process in vitroand in vivo thereby having a very significant repercussion in fibrosisdevelopment, and demonstrate that this reduces the amount of fibrosis inthe pulmonary parenchyma.

All of the patents and publications referred to herein are incorporatedherein by reference in their entirety, as if fully set forth herein.

From the foregoing, it can be seen that the present application providesadvances over the art in the treatment of pulmonary fibrosis, includingIPF. These advances include, without limitation those reflected in thefollowing claims:

The invention claimed is:
 1. A method for treatment of pleural orpulmonary fibrosis in a subject in need of such treatment comprisingadministering to the subject means for inhibiting heat shock protein 27(HSP27).
 2. The method of claim 1, wherein the pleural or pulmonaryfibrosis is idiopathic pulmonary fibrosis (IPF).
 3. The method of claim1, wherein the pleural or pulmonary fibrosis is subpleural fibrosis,Usual Interstitial Pneumonia (UIP), or drug-induced lung fibrosis. 4.The method of claim 1, wherein the means for inhibiting HSP27 isadministered by way of inhalation.
 5. The method of claim 1, wherein themeans for inhibiting HSP27 is administered by way of intra-pleuralinjection, intravenous injection, or intra-tracheal injection.
 6. Themethod of claim 1, wherein the subject is human.
 7. The method of claim6, wherein the pleural or pulmonary fibrosis is idiopathic pulmonaryfibrosis (IPF).
 8. The method of claim 6, wherein the pleural orpulmonary fibrosis is subpleural fibrosis, Usual Interstitial Pneumonia(UIP), or drug-induced lung fibrosis.